Isolation and sequence of cDNA clones coding for a member of the family of high mobility group proteins (HMG-T) in trout and analysis of HMG-T-mRNA''s in trout tissues.

A specific oligonucleotide has been used to isolate a cDNA prepared from the mRNA for a trout High Mobility Group (HMG) protein closely related to trout HMG-T and bovine HMG 1 and 2 proteins. The sequence isolated more closely resembles bovine HMG-1 than the previously sequenced HMG-T protein in regions corresponding to the N terminal half of the protein. Northern blot analysis at low stringency indicated that 2 related sequences are expressed in total trout testis mRNA. Southern blots of total trout DNA indicate that several different forms of the homologous sequence are present in the trout genome and an estimate of copy number by dot-blot shows 4 HMG-T genes per trout sperm DNA equivalent. Analysis of mRNA from several trout tissues, including testis, liver and kidney indicates that expression of genes for histones and the larger HMG proteins in trout is not closely coupled.     

Isolation and in vitro translation of a mammalian protamine mRNA

mRNA was isolated from sexually mature rat, rabbit, and bovine testes. Poly(A+) and (A-) RNAs were prepared and hybridized to a rainbow-trout protamine probe. The bovine (A+) fraction showed significant hybridization compared to the other species and these related sequences were also found in total bovine DNA. Bovine mRNA programmed the in vitro synthesis of a basic protein that possessed protamine-like properties. The mRNA was fractionated by agarose-gel electro-phoresis and the fractions hybridized to the trout protamine probe. A significant hybridization signal was observed corresponding to a mRNA of approximately 400 nucleotides in length which coded for the protamine-like protein. The data support the view that we have isolated a mammalian (bovine) protamine mRNA.     

Divergence of protamine gene sequences in fish

Complementary DNA to trout protamine mRNA was hybridized to excess genomic DNA from trout, salmon and yellow perch. Although there was extensive hybridization of the cDNA to trout DNA, no cross-reaction with yellow perch DNA was observed and the hybridization to salmon DNA was noticeably less than in the homologous reaction. To confirm these results, yellow perch protamine mRNA was purified and compared directly to trout protamine mRNA. Yellow perch protamine mRNA was shorter than trout protamine mRNA, when measured by agarose gel electrophoresis in the presence of methyl mercury hydroxide. The two mRNAs did not cross-react in cDNA/RNA hybridizations, although the homologous reactions went to 90% of completion. This lack of sequence homology was confirmed when the oligopyrimidine tracts from the cDNAs were compared. No sequences longer than tetranucleotides were common to both species. Trout protamine cDNA contained oligopyrimidines of composition C₇T₄, C₄T₂, C₃T₂, C₂T₄, C₂T₃, C₁T₅ and C₁T whereas yellow perch protamine cDNA contained C₆T₃ and C₄T₃.     

Molecular analysis of the protamine multi-gene family in rainbow trout testis.

We have synthesized a family of double-stranded cDNAs (ds cDNAs) using as a template the family of highly purified protamine mRNAs from rainbow trout testis. Individual pure protamine cDNA components were isolated by cloning this family of protamine ds cDNAs in a plasmid vector (pMB9). Clones containing protamine sequences were characterized by restriction mapping and by a positive hybrid-selected translation assay, which allowed us to correlate particular cDNAs with particular protein components. To allow more detailed comparisons, complete nucleotide sequences were determined for selected protamine clones. We have detected at least 5 distinctly different coding sequences, which nevertheless show at least 82% homology, and which have probably arisen by repeated gene duplication. These very highly conserved coding sequences do however contain a distinctly variable region near the 5'-end of the mRNA (N-terminus of the protein), corresponding to the major sites of serine phosphorylation. Since the amino acid sequences predicted by our DNA sequences were slightly different from those previously published (1), we have independently determined the amino acid sequences of protamine components CI, CII, CIII from our own source of trout testis. These new peptide sequences are completely consistent with those predicted by our nucleotide sequences. The 3'-untranslated regions of the protamine mRNAs are, surprisingly almost as highly conserved as the coding regions. Both coding and 3'-noncoding portions appear to be under a similar degree of selective pressure and evolutionary constraint to remain constant.     

Binding of a phosphoprotein to the 3'' untranslated region of the mouse protamine 2 mRNA temporally represses its translation.

The synthesis of the protamines, the predominant nuclear proteins of mammalian spermatozoa, is regulated during germ cell development by mRNA storage for about 7 days in the cytoplasm of differentiating spermatids. Two highly conserved sequences, the Y and H elements present in the 3' untranslated regions (UTRs) of all known mammalian protamine mRNAs, form RNA-protein complexes and specifically bind a protein of 18 kDa. Here, we show that translation of fusion mRNAs was markedly repressed in reticulocyte lysates supplemented with a mouse testis extract enriched for the 18-kDa protein when the mRNAs contained the 3' UTR of mouse protamine 2 (mP2) or the Y and H elements of mP2. No significant decrease was seen when the fusion mRNAs contained the 3' UTR of human growth hormone. The 18-kDa protein is developmentally regulated in male germ cells, requires phosphorylation for RNA binding, and is found in the ribonucleoprotein particle fractions of a testicular postmitochondrial supernatant. We propose that a phosphorylated 18-kDa protein plays a primary role in repressing translation of mP2 mRNA by interaction with the highly conserved Y and H elements. At a later stage of male gamete differentiation, the 18-kDa protein no longer binds to the mRNA, likely as a result of dephosphorylation, enabling the protamine mRNA to be translated.     

Protamine messenger RNA: partial purification and characterization of a heterogeneous family of polyadenylated messenger components.

Poly(A)+ protamine mRNA (pmRNA) components were isolated after separation on denaturing preparative polyacrylamide gels. The four size classes of protamine mRNA described previously were found to contain poly(A) tracts of different lengths. The pmRNA1 was found to be associated with (A)110, pmRNA2 with (A)90, pmRNA3 with (A)85, and pmRNA4 with (A)69. Following deadenylation with RNase H after duplex formation with oligo-dT, the isolated mRNAs were found to be still heterogeneous, although highly enriched in certain of the deadenylated components. DNA complementary to the isolated mRNAs (cDNA) was synthesized in vitro. Following depurination, the oligopyrimidine maps indicated that C7T4, corresponding to an Arg-Arg-Gly-Gly sequence in protamine and originally thought to be characteristic of all mRNA components, is present in only one or possibly tow of the components. Cross-hybridizations between the cDNAs and the four poly(A)+ pmRNAs indicated that a basic polynucleotide unit of substantial length is common to all four mRNAs and that the existing nucleotide sequence variations probably originate from one or both of the non-coding portions of the mRNA molecules.     

Heterogeneity of zein mRNA and protein in maize

Zein, the prolamine fraction of maize, is localized in the endosperm in membrane-bound structures called protein bodies, which have polyribosomes on their surfaces. These polysomes or the mRNA fraction isolated from them will direct the synthesis of zein-like proteins in vitro. The in vitro products consist primarily of two molecular weight classes but show considerable charge heterogeneity when analyzed by isoelectric focusing. Although the molecular weight classes are very similar for different inbred lines, the isoelectric focusing patterns differ.Results given here suggest that the extensive charge heterogeneity of zein proteins does not result from the presence of a large number of totally distinct mRNAs. Zein proteins synthesized in vitro fall into several families related by sequence homologies in their mRNAs. In Illinois High Protein (IHP) the major zein mRNAs can be classified into three families based on their binding to cloned complimentary DNA copies of IHP zein mRNA. Each of three other lines we have studied (W22, Oh43, and W64A) has zein mRNAs that are related to those of IHP. Among these four lines the molecular weights of the members of a given family are generally similar, but the number of members in a family and their isoelectric points differ.     

Cloning of Protamine cDNA of the Medaka (Oryzias latipes) and Its Expression during Spermatogenesis

Protamines or sperm specific basic proteins are highly basic low molecular weight proteins that substitute histones in the chromatin of sperm during spermatogenesis. They condense sperm DNA into a highly compact, stable and inactive complex. In this study, cDNA of protamine of the medaka, Oryzias latipes , was cloned to elucidate the molecular mechanisms involved in spermatogenesis. A medaka testis cDNA library constructed in lambda gt 11 showed 2.78X10⁶ independent recombinants. Several positive clones were obtained by immunoscreening with polyclonal antiserum against medaka protamine. Sequencing showed that one of these positive clones, named MP-1, encoded arginine clusters characteristic of protamine. The putative amino acid sequence of MP-1 revealed a remarkable extent of homology with other fish protamines, such as 71% identity with thynnin Y, a sperm specific basic protein isolated from the bluefin tuna, Thunnus thynnus . Northern hybridization using a MP-1 cDNA probe showed that MP-1 mRNA is present exclusively in the testes and that it gave three detectable bands: a major band of 280 b, and two others of 400 b and 500 b. In situ hybridization of a complementary RNA probe (digoxigenine-UTP-labeled MP-1 RNA) revealed that MP-1 mRNA is localized in some secondary spermatocytes and spermatids, but not in primary spermatocytes or spermatogonia. These results differ from those obtained in studies on the rainbow trout by solution hybridization and in situ hybridization.     

DNA amplification in antifolate-resistant Leishmania. The thymidylate synthase-dihydrofolate reductase gene and abundant mRNAs

Leishmania tropica promastigotes selected for resistance to the dihydrofolate reductase inhibitor, methotrexate, or the thymidylate synthase inhibitor, 5,8-dideaza-10-propargyl folate, overproduce a bifunctional thymidylate synthase-dihydrofolate reductase and possess a 30-kilobase region of amplified DNA. Five fragments, resulting from BglII digestion of this amplified DNA, were cloned into vectors and utilized as probes to examine mRNA in these organisms. Four mRNA species which hybridize to the amplified DNA sequences were found in both resistant and wild-type Leishmania, but were about 40-fold more abundant in the drug-resistant cells. Three of the four mRNAs are transcribed from the same strand of DNA, are clustered, and appear to have partial overlapping sequences. The thymidylate synthase-dihydrofolate reductase gene was localized to a specific region of the amplified unit of DNA by hybridization with mouse cDNA containing thymidylate synthase sequences and with a synthetic oligonucleotide 41 nucleotides in length, prepared on the basis of the partial amino acid sequence of the Leishmania enzyme. Furthermore, mRNA hybrid-selected using a plasmid containing sequences of the putative gene was shown to direct in vitro synthesis of the bifunctional protein.     

Isolation of a cloned DNA segment containing a ribosomal protein gene of Drosophila melanogaster

A Drosophila genomic DNA library in the vector Charon 4 was screened using cDNA derived from the small (6S-12S) poly(A)+ mRNA of 2-6-h-old Drosophila embryos. This fraction of mRNA is enriched for ribosomal protein-coding sequences. The selected recombinants were hybridized to total mRNA under conditions which allowed for isolation of homologous mRNAs. The mRNA from these RNA/DNA hybrids was eluted and translated in vitro. The translation products were analyzed by one- and two-dimensional electrophoresis with authentic ribosomal proteins as standards. One cloned DNA segment was found to contain a ribosomal protein gene, and a sequence which hybridizes strongly to at least 5 other ribosomal protein mRNAs.     

Isolation and characterization of trout testis protamine mRNAs lacking poly (A)

Poly(A)+ protamine mRNA was isolated from trout testis cells in a very pure form, and artificial poly(A)- protamine mRNA molecules were derived from it by enzymatic deadenylation with RNAase H from calf thymus after hybridization with oligo(dT). The deadenylated protamine mRNA was found to be active in a wheat germ cell-free system and yielded a labeled product which co-migrated with authentic protamine. These deadenylated mRNA molecules were subsequently used as markers on denaturing polyacrylamide gels to identify and allow the purification of the poly(A)- protamine components known to exist in vivo in the total cellular poly(A)- RNA. RNA species of molecular weights similar to the enzymatically deadenylated subcomponents of protamine mRNA were observed in the natural poly(A)-RNA population of the testis cells. These naturally occurring poly(A)- protamine mRNAs were isolated by preparative gel electrophoresis and further characterized by 3H-poly(U) hybridization assay, by hybridization to complementary DNA made against highly purified poly(A)+ protamine mRNA, and by their ability to direct protamine synthesis in a cell-free system.     

The envelope-associated 22K protein of human respiratory syncytial virus: nucleotide sequence of the mRNA and a related polytranscript

We recently determined that respiratory syncytial virus (strain A2) encodes a fourth unique envelope-associated virion protein that has molecular weight of approximately 24,000, as estimated by gel electrophoresis. The nucleotide sequence of the mRNA encoding this novel protein has now been determined from five cDNA clones, including three that contain the complete mRNA sequence. The complete mRNA sequence is 957 nucleotides, exclusive of polyadenylate, and contains two partially overlapping open reading frames. The 5'-proximal open reading frame is favored for utilization by the criteria of the location and sequence of its translational start site. Furthermore, the calculated molecular weight of the encoded protein, 22,153, is in agreement with the previous estimate of 24,000 for the authentic protein identified by hybrid selection and in vitro translation. The sequence of the predicted protein, now designated the 22K protein, contains 194 amino acids, is relatively hydrophilic, and appears to be the most basic of the respiratory syncytial virus proteins. The mRNA also contains a second, internal open reading frame which would encode a protein of 90 amino acids. However, no evidence for this translation product is known. The first nine nucleotides in the mRNA sequence, 5'-GGGGCAAAU, are identical to the conserved sequence identified previously at the 5' termini of seven other respiratory syncytial viral mRNAs; the sequence at the 3' end of the 22K mRNA, 5'. . . AGUUAUUU-polyadenylate, contains the elements of the previously identified 3'-terminal consensus sequence for respiratory syncytial virus mRNAs, AGUUAA(N)1-4-polyadenylate (P. L. Collins, Y. T. Huang, and G. W. Wertz, Proc. Natl. Acad. Sci. U.S.A. 81:7683-7687). In addition, we present and describe the intergenic sequence of a dicistronic RNA derived from readthrough of the F and 22K protein genes.     

Chick tropoelastin isoforms. From the gene to the extracellular matrix

Studies from several laboratories have demonstrated the existence of multiple tropoelasting mRNAs and protein isoforms. The present study was designed to examine the developmental expression of a specific tropoelastin mRNA, its encoded isoform, and the fate of that isoform in the extracellular matrix. A chick genomic DNA library was screened with a chick tropoelastin cDNA. Seven unique, overlapping clones spanning 39 kilobases were isolated. A synthetic oligonucleotide complementary to a variable tropoelastin mRNA sequence was used to identify a 1.5-kilobase PstI-BamHI genomic fragment. Nucleotide sequence data revealed that the putative exon was surrounded by intron sequences possessing canonical splice sites at the exon/intron borders. Using both immunologic and molecular probes specific to the tropoelastin isoform and mRNA, quantitative protein and RNA analyses were performed. Results demonstrate that total tropoelastin mRNAs increased significantly during aortic embryogenesis whereas the amount of mRNA containing the variable exon remained relatively constant. The amount of total tropoelastins within the same developmental period reflect the level of total tropoelastin mRNA. The amount of the tropoelastin isoform containing the variable exon essentially mirrored the corresponding mRNA with the exception that a decrease in the isoform at day 15 was not seen in the mRNA level. Immunoelectron micrographs of 13-day chick aortic tissue using both total and isoform-specific antisera showed ultrastructural localization to definable elastic fibers. Antibodies to the variable tropoelastin isoform occurred preferentially at sites where elastic fiber microfibril structures were evident.     

RNA-binding properties and translation repression in vitro by germ cell-specific MSY2 protein

The large amount of MSY2 protein, a mouse germ cell-specific Y-box protein, in oocytes and its degradation by the late two-cell stage suggest that MSY2 may stabilize and/or regulate the translation of maternal mRNAs. We report here the ability of bacterially expressed recombinant MSY2 protein to bind to mRNA and repress translation in vitro. Although MSY2 displays some sequence specificity in binding to short RNA sequences derived from the 3' untranslated region (UTR) of the protamine 1 (Prm1) mRNA, as determined by both gel shift and filter binding assays, essentially no sequence specificity is observed when full-length Prm1 mRNA is used. The binding of MSY2 is approximately 10-fold greater to the full-length Prm1 mRNA than to a 37-nucleotide sequence derived from the 3' UTR, and gel shift assays indicate that multiple MSY2 molecules bind to a single Prm1 mRNA. MSY2 binding to luciferase mRNA at ratios of protein to mRNA that are likely to exist in the oocyte also leads to a moderate inhibition of protein synthesis in vitro. Given the abundance of MSY2 in mouse oocytes (2% of total oocyte protein), these data suggest that MSY2 packages mRNAs in vivo with relatively little sequence specificity, which may lead to both stabilization and translation repression of maternal mRNAs.     

Divergent RNA binding specificity of yeast Puf2p

PUF proteins bind mRNAs and regulate their translation, stability, and localization. Each PUF protein binds a selective group of mRNAs, enabling their coordinate control. We focus here on the specificity of Puf2p and Puf1p of Saccharomyces cerevisiae, which copurify with overlapping groups of mRNAs. We applied an RNA-adapted version of the DRIM algorithm to identify putative binding sequences for both proteins. We first identified a novel motif in the 3' UTRs of mRNAs previously shown to associate with Puf2p. This motif consisted of two UAAU tetranucleotides separated by a 3-nt linker sequence, which we refer to as the dual UAAU motif. The dual UAAU motif was necessary for binding to Puf2p, as judged by gel shift, yeast three-hybrid, and coimmunoprecipitation from yeast lysates. The UAAU tetranucleotides are required for optimal binding, while the identity and length of the linker sequences are less critical. Puf1p also binds the dual UAAU sequence, consistent with the prior observation that it associates with similar populations of mRNAs. In contrast, three other canonical yeast PUF proteins fail to bind the Puf2p recognition site. The dual UAAU motif is distinct from previously known PUF protein binding sites, which invariably possess a UGU trinucleotide. This study expands the repertoire of cis elements bound by PUF proteins and suggests new modes by which PUF proteins recognize their mRNA targets.